International Research Journal of Microbiology Vol. 2(4) pp. 116-121, May 2011
Available online http://www.interesjournals.org/IRJM
Copyright © 2011 International Research Journals
Full Length Research Paper
Production of L-Glutamic acid by Corynebacterium
glutamicum DSM 20300T and Arthrobacter globiformis
MTCC 4299 using fruits of Muntingia calabura Linn.
Payala Vijayalakshmi*, Dhurjeti Sarvamangala
* PhD Research scholar in Gitam institute of sciences, Gitam University, Rushikonda, Visakhapatnam-530 045 (Andhra
Pradesh), INDIA
Asst.Prof. in Gitam institute of technology Gitam university, Rushikonda, visakhapatnam-530 045(A.P)
Accepted 20 May, 2011
The present study was an illustrative investigation on L-Glutamic acid production by using two
different bacterial strains like Corynebacterium glutamicum DSM 20300T and Arthrobacter
globiformis MTCC 4299 employing a cheaply available fruit substrate of Muntingia calabura L.
Owing to the high sugar content of these fruits and quick establishment process of its plant an
attempt was made to utilize the fruits for the production of glutamic acid, the precursor of mono
sodium glutamate (MSG), employing submerged fermentation. In these experiments, optimization
of fermentation parameters could be done using an empirical strategy method to determine key
factor ranges for the production of glutamic acid. After optimization of physicochemical
parameters, the highest production of glutamic acid was obtained 15.1 mg/ml with
Corynebacterium glutamicum DSM 20300T at pH 7.0, temperature 300C was maintained for 48h
incubation time with urea, biotin and penicillin optimum concentration were being 2.0g/l, 1µg/l and
1U/ml respectively. On the other hand, the production of glutamic acid obtained with Arthrobacter
globiformis MTCC 4299 was 10.7mg/ml at pH 7.0, temperature 300C time 48 h, urea 2.0g/l, biotin
4µg/l and penicillin concentration 15U/ml. Agitation at a speed of 120 rpm was favourable for the
production. Eventually the product which was produced was analyzed by thin layer
chromatography and subjected to crystallization at its isoelectric pH 3.2. Finally the product
presence was confirmed by Liquid chromatography-Mass spectroscopy.
Keywords: Muntingia calabura L., Corynebacterium glutamicum, Arthrobacter globiformis, Glutamic acid,
Fermentation and Crystallization.
INTRODUCTION
Glutamic acid is of industrial importance because; it is
widely used in food, pharmaceutical, medical,
*Corresponding author E-mail: bavisettyvijayalakshmi2@gmail.com;
Tel: 91-9966068683
Abbreviations
TLC: Thin layer chromatography
Rf: Retardation factor
Triple QQQ LC/MS: Triple Quadrupole Liquid chromatography/Mass
spectroscopy.
M.calabura: Muntingia calabura
C.glutamicum: Corynebacterium glutamicum
A.globiformis: Arthrobacter globiformis
biochemical and analytical industries. Mono sodium
glutamate (MSG), the sodium salt of glutamic acid is used
commercially as a flavor enhancer, usually in combination
with nucleotides inosinate to provide an expansion and
extension of taste in processed foods. Besides the
existence of efficient methods of industrial fermentation,
many efforts are still on to improve the glutamic acid
fermentation, particularly under the stand point of
cheaper available substrates to help go down the
production costs.
In the past few years, L-glutamic acid has been
synthesized from a variety of raw materials through
submerged fermentation. Literature survey revealed that,
glutamic acid was produced from palm waste hydrolysate
Vijayalakshmi and Sarvamangala 117
supplemented with glucose using Brevibacterium
lactofermentum ATCC 13869 Das et al. (1995). Several
other agro waste materials were also used for the
production of glutamic acid cassava starch Jyothi et al.
(2005), sugar cane baggase (Amin and Al-Talhi, 2007)
under submerged fermentation conditions. Later on, an
investigation carried out by Mahmoud Tavakkoli et al.
(2009) reported that a high amount of glutamic acid was
excreted from date waste juice by Corynebacterium
glutamicum CECT 690. Response surface methodology
was used to optimize the submerged fermentation
conditions. Glutamic acid can also be produced through
solid state fermentation process (SSF). Sugar cane
baggase enriched with 10%glucose liberated glutamic
acid 80mg/g of dry sugar cane baggase (Nampoothiri and
Pandey, 1996). However submerged fermentation was
commonly used for the production of glutamic acid. The
present method differed from all the other methods which
had been reported so far, in that the fruit pulp of
Muntingia calabura L. ( M.calabura L.) was used as
carbon source inorder to get very higher yields of
glutamic acid. The plant M.calabura L. (Elaeocarpaceae)
usually known as Jamaica cherry or panama berry is a
fruit species indigenous to Southern Mexico, Central
America, Northern South America and the Greater
Antilles, that has been distributed in south-east Asia and
many other places as a road side adornment. In tropical
lowlands it can sustain continuous growth. It tends to be
an invasive plant since birds disperse the seeds and they
germinate readily. The fruits have high nutrient value and
rich in carbohydrate proportion (Janick and Paull, 2008).
Tonnes of these fruits are being wasted or unutilized and
containing a richest amount of sugars so conversion of
these fruits to a useful product like glutamic acid is a
novel thought.
One of the main intent of the current research was to
optimize the basal nutrients and fermentation parameters
like urea, biotin and penicillin concentrations, pH,
temperature and time for getting high glutamic acid
production using empirical optimization strategy or
traditional ‘one-factor-at-a-time’ method. This involves
varying one factor while keeping the other factors
unchanged under a specific set of conditions.
Selection of suitable microorganisms is one of the
most important stages of any fermentation process. In the
present study, two different bacterial cultures like
Corynebacterium glutamicum (C.glutamicum) and
Arthrobacter globiformis (A.globiformis) have been
chosen as they are high yielding strains of glutamic acid,
biotin dependent and capable of growing in a natural
media. Both are gram positive rods and produce white
colour colonies on nutrient agar.
MATERIALS AND METHODS
Microorganisms
The bacterium Corynebacterium glutamicum DSM
T
20300 was obtained from the National chemical
laboratory, Pune, India and Arthrobacter globiformis
MTCC 4299 was procured from IMTECH, Chandigarh
and were used in this study. The bacterial cultures were
maintained on a basal nutrient agar slopes and stored in
a refrigerator at 40C.
Medium used
The ripened fruits of M.calabura which contain rich
quantity of sugars used to compensate the basic carbon
requirements of the organisms. Urea was sterilized
separately by filtration with Seitz filter and then added to
the basal medium to supply the nitrogen needs of the
culture. Other mineral salts K2HPO4, KH2PO4, MgSO4,
MnSO4, Fe2 (SO4)3, biotin, penicillin were sterilized
separately by autoclaving at 1210C for 15 minutes and
added to the production medium.
Cultural conditions
10g of fruit pulp was dispensed separately in 250ml
Erlenmeyer flasks containing 100ml of distilled water and
mineral salts (urea: 0.8g/l, K2HPO4 :0.08g/l, KH2PO4:
0.08g/l , MgSO4: 0.04g/l ,Mnso4:001g/l , Fe2(SO4)3 :
0.001g/l, Biotin: 1µg/l) and 1% of inoculum was added to
each flask and after 24h of fermentation an aqueous
solution of crystalline penicillin G was added at a
concentration of 1U/ml. However the addition of penicillin
prior to 24h of fermentation would inhibit the production.
The fermentation was carried out at varying physical
conditions like temperature (20, 25, 30, 35, 40) pH (5.0,
6.0, 7.0, 8.0, 9.0), time (24, 48, 72, 96, 120h) and
chemical conditions like urea concentration (0.8, 1.2, 1.6,
2.0, 2.4 g/l), biotin concentration (1, 2, 3, 4, 5 µg/l) and
H
penicillin concentration (1, 5, 10, 15, 20 U/ml). P
adjustment was done by using 1N HCl or 1N NaOH.
Analytical methods
Estimation of sugars in the fruit substrate
Preliminarily 100 mg of the fruit sample was weighed and
subjected to acid hydrolysis with 5ml of 2.5 N Hcl for 3h.
118 Int. Res. J. Microbiol.
Later, the cooled hydrolysate was neutralized with
Na2CO3 until the effervescence completely ceases and
was subjected to centrifugation by adjusting the volume
to 100ml. Subsequently the resultant supernatant was
separated and subjected to various qualitative and
quantitative tests for determination of sugars. Phenol
sulphuric acid method Dubois et al. (1956) was used for
the quantitative determination of sugars in the fruit
sample.
Determination of glutamic acid
The fermented broth at the end of fermentation was
subjected to centrifugation at 10,000×g for 10 minutes to
remove microbial cells. The collected supernatant was
later subjected to thin layer chromatography (TLC) for
qualitative identification of glutamic acid by using a
solvent mixture containing 1-butanol, acetic acid, water in
80/20/20 (v/v) and adsorbent silica Gel G and running
standard amino acids simultaneously on a chromatogram
(Sadasivam and Manickam, 2005). The concentration of
glutamic acid was quantitatively estimated by standard
ninhydrin method (Sasidhar Rao and Vijay Deshpande,
2005). The product was finally confirmed by Agilent 6410
Triple
Quadrupole
Liquid
chromatography-mass
spectroscopy (Agilent 6410 QQQ LC/MS) was used
because it analyze the target compounds in complex
matrices. In the mass spectra the desired compound
produces a molecular ion peak located at m/z
corresponding to its molecular mass. Agilent 6410
QQQLC/MS utilize advanced nebulization technology and
have three consecutive quadrupoles arranged in series to
incoming ions.
The test sample for LC/MS was obtained by
subjecting the fermented broth to crystallization at
isoelectric pH 3.2 using 1N Hcl and eventual drying in a
vacuum desiccator yielded crystals of L-glutamic acid.
The resultant granular crystals were visualized through
microscopy.
RESULTS AND DISCUSSION
Glutamic acid has been the object of scientific study for
over 150 years and has been sold as chemical product
for the last eight decades. The highest produced amino
acid approximately 9, 00,000 tonnes per year is glutamic
acid which clearly demonstrates a proof of its increasing
demand. Besides the existence of efficient methods of
industrial fermentation, many efforts are still on to
improve the glutamic acid fermentation, particularly under
the stand point of cheaper available substrates to help go
down the production costs. In the past few years, an
elaborative investigation has been done to establish the
most pertinent and economically durable raw material for
glutamic acid production.
A variety of cheapest saccharine materials like
molasses, cane juice, beet juice, cassava roots, sago etc.
are used for the production of glutamic acid Kono et al.
(1965). Later on a research is carried out by Das et al.
concludes that, palm waste hydrolysate is the best
substrate which gives an elevated concentration of
glutamic acid 88g/l using submerged fermentation.
Despite, the existence of number of raw materials which
fit into the above mentioned category, the present study
introduces another novel substrate for glutamic acid
fermentation- fruits of M. calabura. The plant M.calabura
L. is an ever green tree found commonly on wastelands
and its fruits become unutilized and left for natural
degradation by soil microbial flora. These fruits were used
as a substrates in the current research and subjected to
glutamic acid submerged fermentation using OFAT
method produces appreciable quantities of product i.e.,
88.8% of glutamic acid higher than the yield acquired by
the earlier studies on glutamic acid production from the
substrates like sugarcane baggase (3.86%) by Jyothi et
al., cassava starch (75.7%) by Amin et al., Mexican lime
waste (13.7%) by Islas-Murguia et al. (1984), biotin rich
beet molasses (55%) by (Haruo Momose and Takashi
Takagi, 1978) , Palm waste hydrolysate (88g/l) by Das et
al. The fruit sample consists of 17.0g of sugars per 100g
of substrate as per the analysis report obtained from
spectrophotometric phenol sulphuric acid method read at
490nm. Nearly 15.1g of sugars out of 17.0g were
converted to glutamic acid. Qualitative sugar analysis
reports of the fruit sample showed the presence of
glucose, fructose and sucrose.
The entire experimental study was performed in
Erlenmeyer flasks under aseptic conditions at varying
physicochemical parameters using empirical strategy for
the determination of the optimal cultural conditions to be
maintained for the large-scale and real time fermentative
production of glutamic acid. Glutamic acid production was
analyzed qualitatively by TLC and quantitatively by
ninhydrin method at various stages of fermentation by
drawing minute samples intermittently during the process.
The result of thin layer chromatography was observed as
a visible purple coloured spot having similar Rf value
(0.33) similar to that of authentic standard sample (0.35).
The yield (%) was calculated as g of glutamic acid
produced per 100g of sugar consumed in the fruit
sample.
The data represented in various Figures 1a - f showed
the production of glutamic acid from the fruits of M.
calabura L. by C.glutamicum and A.globiformis at varied
fermentation conditions.
Vijayalakshmi and Sarvamangala 119
5
C.glutamicum
0
A.globiformis
5
6
7
8
7
conc. of glutamic acid(g/l)
conc. of glutamic
acid(g/l)
8
10
9
6
5
4
3
C.glutamicum
2
A.globiformis
1
0
pH
0.8
1.2
1.6
2
2.4
urea concentration (g/l)
a
H
Figure1 : Effect of p on glutamic acid production. Indicates that
the optimum concentration of glutamic acid 5.88mg/ml for
C.glutamicum and 5.28mg/ml for A.globiformis was recorded at
H
p 7.0
d
Figure1 : Effect of urea concentration on glutamic acid
production. Indicates that the concentration of urea 2% (W/V)
leads to maximal concentration of glutamic acid 4.20mg/ml and
7.08mg/ml by C.glutamicum and A.globiformis.
5
4
3
2
C.glutamicum
1
A.globiformis
0
24
48
72
96
120
conc. of glutamic acid(g/l)
conc. of glutamic acid(g/l)
6
4
3.5
3
2.5
2
1.5
1
0.5
0
C.glutamicum
A.globiformis
1
Time (h)
2
3
4
5
Biotin concentration (µg/l)
b
e
Figure1 : Effect of biotin concentration on glutamic acid
production. Shows the effect of biotin concentration on glutamic
acid production 3.44mg/ml from C.glutamicum and 3.72mg/ml
from A.globiformis with optimum biotin concentration 1 µg/l and
4µg/l.
conc.of glutamic acid(mg/ml)
Figure1 : Effect of time on glutamic acid production.
Maximum production 3.92mg/ml and 5.12mg/ml was obtained
with C.glutamicum and A.globiformis at 48h of fermentation with
different time intervals.
16
14
12
10
8
6
4
2
0
C.glutamicum
A.globiformis
1
5
10
15
20
penicillin concentration(U/ml)
f
c
Figure1 : Effect of temperature on glutamic acid production.Shows
the effect of temperature on optimum production of glutamic acid
o
4.04mg/ml and 2.64mg/ml by C.glutamicum and A.globiformis at 30 c
temperature.
Effect of pH
The influence of pH was studied carefully on the yield of
glutamic acid by subjecting the fermentation flasks to N
varying pH values ranging from 5.0-9.0 by the addition IN
Figure1 : Effect of penicillin concentration on glutamic acid
production. Penicillin was one of the major critical factors on Lglutamic acid production. Maximum concentration of glutamic acid
14.3mg/ml by C.glutamicum and 9.8mg/ml by A.globiformis was
achieved when the penicillin was added at a concentration of 1
U/ml and 15U/ml to the production medium
IN Hcl/1N NaoH. Consequently it was observed that pH
7.0 favours the maximum yield of glutamic acid in both
C.glutamicum (34.5%) and A.globiformis (31.05%). The
production was also tested from pH 7.1-7.9 but the
H
readings were declined beyond p 7.0
120 Int. Res. J. Microbiol.
Figure 2: Mass spectrum of glutamic acid with C.glutamicum culture fermentation
Effect of time
The fermentation process was allowed to operate till 120h
and the broth was periodically examined every 24h for
maximum production. Fermentation efficiency of C.
glutamicum (23.05%) and A.globiformis (30.1%) was
observed to be high at 48h which gradually decreased
upon further incubation.
Effect of temperature
Optimum temperature was a pre requisite for the opulent
yield of glutamic acid. So, initially the production was
tested under various incubation temperatures. In case of
both the organisms, 300C were found to be the ambient
temperature yielding (23.7%) and (15.5%) for
C.glutamicum and A. globiformis respectively.
Effect of urea
Urea was found to be the best and cheapest nitrogenous
source for glutamic acid production. So, the study
included determination of appropriate concentration of
urea which gave highest output of glutamic acid
production. A concentration of 2.0 g/l of urea was found
to be suitable for the fermentation process yield (24.7%)
with C.glutamicum and (41.6%) with A.globiformis.
Effect of Biotin
Surplus production of glutamic acid by microorganisms
was found to be due to the permeability change induced
by limiting the supply of biotin required by the bacterium.
The concentration of biotin must be suboptimal for
growth. Biotin in very low amount prevents the growth of
microorganisms and high level inhibits L-glutamic acid
production by feedback control. Biotin concentration of
1µg/l and 4µg/l were optimum for the excretion of
glutamic acid (20.2%) and (21.8%) by C. glutamicum and
A. globiformis.
Effect of penicillin
Owing to its lytic activity on the cell wall there by
releasing glutamic acid into medium, penicillin was found
to be the most critical factor in the cellular excretion of
glutamic acid. Penicillin was added at frequent intervals
of 6h till 48h to fermentation flasks. But the Maximum
yield of glutamic acid was obtained 84.1% with C.
glutamicum and 57.6% with A. globiformis by the addition
of penicillin to the production medium only after 24h of
fermentation at a concentration of 1U/ml to C.glutamicum
and 15U/ml to A.globiformis.
After
standardizing
all
the
physicochemical
parameters at optimum conditions of production (pH7.0,
time48h, temp.30oC, urea2.0g/l, biotin1.0µg/l and
penicillin1.0U/ml by C.glutamicum and pH7.0, time48h,
0
temp.30 C, urea2.0g/l, biotin4.0µg/l and penicillin15U/ml
by A.globiformis) the fermentation was done again to
achieve maximum production of glutamic acid. The
maximum yield obtained was 15.1 mg/ml (88.8%) with
C.glutamicum and 10.7mg/ml (62.9%) with A.globiformis.
So the present results revealed that significantly high
quantity of glutamic acid was obtained from C.glutamicum
fermentation.
Afterward the finished broth was exploited for
H
obtaining glutamic acid crystals by adjusting the p to
isoelectric point of glutamic acid 3.2. Agilent 6410 QQQ
LC/MS is used for the determination of molecular mass of
glutamic acid. In the resultant two different mass spectra
the molecular ion peaks were located at 149.1(Figure.2)
for C.glutamicum and 148.9 (Figure.3) for A.globiformis
which confirmed the presence of glutamic acid. More
peaks were visible larger than the molecular ion peak due
to solvent used or impurity or an isotope of the molecular
Vijayalakshmi and Sarvamangala 121
Figure 3: Mass spectrum of glutamic acid with A.globiformis culture fermentation
ion or may be due to the presence of more than one
carbon atom in a compound. However peaks with mass
less than the molecular ion were the result of
fragmentation of the molecule. The mass spectroscopy of
the samples was done at [Dept. of organic chemistry,
Andhra University, Visakhapatnam].
CONCLUSION
The organic carbon source in any fermentation processes
is the most expensive component contributing to the cost
of the process. In the present study, the carbon source is
provided by the sugars present in the fruits of Muntingia
calabura Linn. an economical raw material produces high
concentration of glutamic acid under optimum physicochemical conditions. The information conveyed in this
study will help in designing various schemes for scale up
level production of L-glutamic acid which may prove to be
an industrial asset.
ACKNOWLEDGMENT
The author would like to thank Dr. D.Sarvamangala
research guide for her extensive guidance in the present
work. Visakha Govt. Degree and P.G college for Women
and Department of organic Chemistry, Andhra University,
Visakhapatnam for their assistance and technical
support.
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